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Ca2+-dependent hormonal stimulation of ciliary activity.

It has been demonstrated that calcium modulates ciliary activity in protozoa and also in epithelial cilia of the mussel gill. Changes in the frequency of ciliary beat in the oviduct of the salamander have also been associated with movement of Ca2 across the cell membrane. However, the control of ciliary activity in mammals is poorly understood and the function of calcium in mammalian cilia and its role in coupling hormonal effects to changes in ciliary activity have been only preliminarily investigated. The evidence presented here suggests that the stimulus-response coupling of the stimulating effect of prostaglandin E2 (PGE2) on ciliary activity of the rabbit oviduct is carried out by release of intracellular Ca2+.

Animals↗

Allozymic heterozygosity and morphological variation in house sparrows.

Several reports have shown that greater heterozygosity (both between individuals and between populations) is associated with lower morphological variance and asymmetry. Most previous work concerned poikilothermic organisms (for example, fish, butterflies, lizards, shellfish, salamanders and plants). Reports concerning two homoiotherms gave conflicting results. The report by Handford on a songbird, Zonotrichia capensis, failed to support the relationship, although these results have been questioned in the literature. Handford suggested that the lack of relationship in Zonotrichia could indicate a fundamental difference between homoiotherms and poikilotherms, reflected in their apparent differences in heterozygosity. However, we report here on electrophoretic and morphometric studies on another songbird species (Passer domesticus), in which the relationship appears to be upheld. We find consistently, among four locality samples, that the class of individuals of greatest allozyme heterozygosity nearly always exhibits the lowest multivariate morphological variance, and the class of greatest homozygosity nearly always exhibits the highest.

Alleles↗

Cyclic GMP can increase rod outer-segment light-sensitive current 10-fold without delay of excitation.

To test the hypothesis that cyclic GMP is the internal messenger coupling rhodopsin activation to membrane excitation in vertebrate rod photoreceptors, we used a novel technique combining measurement of membrane currents of isolated salamander rods with a suction electrode and the introduction of cyclic GMP through a whole-cell recording patch pipette. Rupture of an attached patch was followed by a rapid (approximately 10 s), approximately 10-fold increase in outer-segment membrane current, all of which was light-sensitive. There was little change in the rising phase of the response to a saturating flash, but the duration of the saturated phase of the response increased approximately 10-fold. The effects reversed completely within 3-4 min after withdrawal of the cyclic GMP-containing patch pipette. A formal kinetic analysis shows that the first two observations are inconsistent with the postulate that cyclic GMP opens the light-sensitive conductance by simple binding to channels, unless free cyclic GMP in the outer segment is assumed to be much lower than published estimates, and most of the outer-segment cyclic GMP is bound and inexchangeable on the timescale of 200 ms. Furthermore, our results suggest that rod cyclic GMP is not involved solely in keeping the light-sensitive conductance open, but may also affect the activity of the phosphodiesterase that mediates cyclic GMP hydrolysis.

3',5'-Cyclic-GMP Phosphodiesterases↗

Cyclic GMP increases photocurrent and light sensitivity of retinal cones.

Like retinal rods, cone photoreceptors contain cyclic GMP and light-activated phosphodiesterase. The cGMP phosphodiesterase cascade is thought to mediate phototransduction in rods. Biochemical assays of nucleotide content in cone-dominant retinas, however, have failed to demonstrate light-induced changes in cGMP. Changes in cyclic AMP following light exposure have been reported, leading to the suggestion that in cone phototransduction cAMP assumes a role analogous to that played by cGMP in rods. Cyclic GMP introduced from tight-seal pipettes into isolated cones of the larval tiger salamander, Ambystoma tigrinum, rapidly increases light-modulated membrane current more than 10-fold. In cones, as in rods, cGMP also causes an approximately 10-fold increase in photocurrent duration and a 5- to 10-fold increase in light-sensitivity. Cyclic AMP has no effect on cone photocurrents under the same conditions. Because cGMP has similar effects on photocurrent magnitude and kinetics in both rods and cones, we conclude that cGMP plays corresponding roles in transduction in both vertebrate photoreceptor classes.

Ambystoma↗

A presynaptic action of glutamate at the cone output synapse.

Neurotransmitter release from many central nervous system synapses is regulated by 'autoreceptors' at the synaptic terminal, which bind the released transmitter and alter release accordingly. The photoreceptors of lower vertebrates are thought to use glutamate as a neurotransmitter. Glutamate conveys the visual signal to postsynaptic bipolar and horizontal cells, but has been reported not to act on the photoreceptors themselves. We show here that glutamate evokes a current, carried largely by chloride ions, in cones isolated from the tiger salamander retina. This response is localized to the synaptic terminal of the cone. Removing external sodium blocks this action of glutamate. These results suggest the existence of a positive feedback loop at the cone output synapse: over most of the light-response range, glutamate released by depolarization of the cone will cause further depolarization, increasing the gain of phototransduction. Glutamate released from rods may also polarize cones, modulating the gain of the cone output synapse. This system is surprisingly different from the autoreceptor systems for most other transmitters, which act in a negative feedback way.

Animals↗

Expression cloning and characterization of a renal electrogenic Na+/HCO3- cotransporter.

Bicarbonate transporters are the principal regulators of pH in animal cells, and play a vital role in acid-base movement in the stomach, pancreas, intestine, kidney, reproductive system and central nervous system. The functional family of HCO3- transporters includes Cl- -HCO3- exchangers, three Na+/HCO3- cotransporters, a K+/HCO3- cotransporter, and a Na+-driven Cl- -HCO3- exchanger. Molecular information is sparse on HCO3- transporters, apart from Cl- -HCO3- exchangers ('anion exchangers'), whose complementary DNAs were cloned several years ago. Attempts to clone other HCO3- transporters, based on binding of inhibitors, protein purification or homology with anion exchangers, have so far been unsuccessful. Here we monitor the intracellular pH and membrane voltage in Xenopus oocytes to follow the expression of the most electrogenic transporter known: the renal 1:3 electrogenic Na+/HCO3- cotransporter from the salamander Ambystoma tigrinum. We now report the successful cloning and characterization of a cDNA encoding a cation-coupled HCO3- transporter. The encoded protein is 1,035 amino acids long with several potential membrane-spanning domains. We show that when it is expressed in Xenopus oocytes, this protein is electrogenic, Na+ and HCO3- dependent, and blocked by the anion-transport inhibitor DIDS, and conclude that it is the renal electrogenic sodium bicarbonate cotransporter (NBC).

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

An accessory chromophore in red vision.

In the absence of a red-sensitive visual pigment, some deep-sea fish use a chlorophyll derivative in their green-sensitive rod cells in order to see deep-red light. Here we show that living rods extracted from a salamander can also accumulate an exogenous chlorophyll derivative, chlorin e6, that renders them as sensitive to red light as they are to green. This vision enhancement by an unbleachable chlorophyll derivative might therefore be a general phenomenon in vertebrate photoreception.

Animals↗

Role of visual pigment properties in rod and cone phototransduction.

Retinal rods and cones share a phototransduction pathway involving cyclic GMP. Cones are typically 100 times less photosensitive than rods and their response kinetics are several times faster, but the underlying mechanisms remain largely unknown. Almost all proteins involved in phototransduction have distinct rod and cone variants. Differences in properties between rod and cone pigments have been described, such as a 10-fold shorter lifetime of the meta-II state (active conformation) of cone pigment and its higher rate of spontaneous isomerization, but their contributions to the functional differences between rods and cones remain speculative. We have addressed this question by expressing human or salamander red cone pigment in Xenopus rods, and human rod pigment in Xenopus cones. Here we show that rod and cone pigments when present in the same cell produce light responses with identical amplification and kinetics, thereby ruling out any difference in their signalling properties. However, red cone pigment isomerizes spontaneously 10,000 times more frequently than rod pigment. This high spontaneous activity adapts the native cones even in darkness, making them less sensitive and kinetically faster than rods. Nevertheless, additional factors are probably involved in these differences.

Animals↗

Recording spikes from a large fraction of the ganglion cells in a retinal patch.

To understand a neural circuit completely requires simultaneous recording from most of the neurons in that circuit. Here we report recording and spike sorting techniques that enable us to record from all or nearly all of the ganglion cells in a patch of the retina. With a dense multi-electrode array, each ganglion cell produces a unique pattern of activity on many electrodes when it fires an action potential. Signals from all of the electrodes are combined with an iterative spike sorting algorithm to resolve ambiguities arising from overlapping spike waveforms. We verify that we are recording from a large fraction of ganglion cells over the array by labeling the ganglion cells with a retrogradely transported dye and by comparing the number of labeled and recorded cells. Using these methods, we show that about 60 receptive fields of ganglion cells cover each point in visual space in the salamander, consistent with anatomical findings.

Action Potentials↗

Spemann's organizer and self-regulation in amphibian embryos.

In 1924, Spemann and Mangold demonstrated the induction of Siamese twins in transplantation experiments with salamander eggs. Recent work in amphibian embryos has followed their lead and uncovered that cells in signalling centres that are located at the dorsal and ventral poles of the gastrula embryo communicate with each other through a network of secreted growth-factor antagonists, a protease that degrades them, a protease inhibitor and bone-morphogenic-protein signals.

Amphibians↗

Activation by mitogens and superantigens of axolotl lymphocytes: functional characterization and ontogenic study.

Urodele amphibians have weak and slow immune responses compared to mammals and anuran amphibians. Using new culture conditions, we tested the ability of lymphocytes of a well-studied salamander, the Mexican axolotl (Ambystoma mexicanum) to proliferate in vitro with diverse mitogenic agents. We demonstrated that the axolotl has a population of B lymphocytes that proliferate specifically and with a high stimulation index to the lipopolysaccharide (LPS) known as a B-cell mitogen in mammals. This proliferative capacity is observed without significant changes throughout ontogenesis. In the presence of LPS, axolotl B lymphocytes are able to synthesize and secrete both isotopes of immunoglobulin described in this species, IgM and IgY. Moreover, a distinct lymphocyte subpopulation is able to poliferate significantly in response to the mitogens usually known as T-cell specific in mammals, phytohaemagglutinin (PHA) and concanavalin A (Con A). The activated cells are T lymphocytes, as shown by depletion experiments performed in vitro with monoclonal antibodies, and in vivo by thymectomy. Splenic T lymphocytes of young axolotls (before 10 months) do not have this functional ability, which suggests maturation and/or migration phenomena during T-cell ontogenesis in this species. Axolotl lymphocytes are able to proliferate in vitro with a significant stimulation index to staphylococcal enterotoxins A and B (SEA and SEB). These products act on mammalian lymphocytes as superantigens: in combination with products of the major histocompatibility complex (MHC), they bind T-cell receptors with particular V beta elements. The fact that these superantigens are able to activate lymphocytes of a primitive vertebrate suggests a striking conservation of molecular structures implied in superantigen presentation and recognition.

Aging↗

Evolutionary genetics of metamorphic failure using wild-caught vs. laboratory axolotls (Ambystoma mexicanum).

In many organisms metamorphosis allows for an ecologically important habitat-shift from water to land. However, in some salamanders an adaptive life cycle mode has evolved that is characterized by metamorphic failure (paedomorphosis); these species remain in the aquatic habitat throughout the life cycle. Perhaps the most famous example of metamorphic failure is the Mexican axolotl (Ambystoma mexicanum), which has become a focal species for developmental biology since it was introduced into laboratory culture in the 1800s. Our previous genetic linkage mapping analysis, using an interspecific crossing design, demonstrated that a major gene effect underlies the expression of metamorphic failure in laboratory stocks of the Mexican axolotl. Here, we repeated this experiment using A. mexicanum that were sampled directly from their natural habitat at Lake Xochimilco, Mexico. We found no significant association between the major gene and metamorphic failure when wild-caught axolotls were used in the experimental design, although there is evidence of a smaller genetic effect. Thus, there appears to be genetic variation among Mexican axolotls (and possibly A. tigrinum tigrinum) at loci that contribute to metamorphic failure. This result suggests a role for more than one mutation and possibly artificial selection in the evolution of the major gene effect in the laboratory Mexican axolotl.

Ambystoma mexicanum↗

Early male reproductive advantage, multiple paternity and sperm storage in an amphibian aggregate breeder.

We tested whether the order in which males encounter females affects reproductive fitness in spotted salamanders (Ambystoma maculatum). Using mating chambers in the field, we allowed one male access to a female before a second male. We then used four microsatellite markers in paternity analyses of the resulting larvae. First males sired a significantly larger number of offspring than second males, suggesting that male reproductive success is greatly enhanced by early arrival at breeding ponds. Multiple paternity was common among clutches, and frequently larvae were assigned to unidentified males that had not been in the chambers. Sperm from these males had either been stored by females for a year or obtained more recently at other breeding sites.

Alleles↗

Molecular systematics and phylogeography of the Plethodon elongatus species group: combining phylogenetic and population genetic methods to investigate species history.

Plethodon elongatus and P. stormi (Caudata: Plethodontidae) are Pacific Northwest endemic species which occur in northwestern California and southwestern Oregon. Studies on these salamanders have resulted in differing taxonomic conclusions, but the underlying historical hypotheses, at both inter- and intraspecific levels, have never been examined in a molecular framework. Here, representatives of 81 populations from throughout the range of both taxa are sequenced. Portions of three mitochondrial protein-coding genes (cytochrome b, NADH dehydrogenase subunit 4, and ATPase 6) were sequenced. Four haplotype groups with nonoverlapping geographical ranges were recovered in separate and combined analyses of the data. One clade corresponds to the distribution of P. stormi, while the remaining three comprise P. elongatus. Phylogenetic relationships among haplotype groups differ in separate analyses of the genes but converge on a well-supported topology, with P. elongatus and P. stormi as monophyletic sister taxa, in combined Maximum Parsimony and Maximum Likelihood analyses. Population genetic analyses of mismatch distributions and Tajima's D-statistic are consistent with range expansion for the largest clade within P. elongatus, covering the northern two-thirds of the species range. In contrast, the P. stormi haplotype clade and the P. elongatus clade from the southern third of the species range may have been relatively stable. Morphological boundaries between P. elongatus and P. stormi are largely congruent with mitochondrial DNA breaks and continued treatment as sister taxa is supported. Although mitochondrial DNA haplotype groups may reflect historical separation within P. elongatus, genetic barriers are incongruent with intraspecific patterns of morphological variation.

Animals↗

Rapid desensitization converts prolonged glutamate release into a transient EPSC at ribbon synapses between retinal bipolar and amacrine cells.

The mechanisms underlying the conversion of prolonged glutamate release from ribbon synapses in bipolar cells to sustained and transient excitatory postsynaptic responses in identified retinal amacrine cells were studied in tiger salamander (Ambystoma tigrinum) retina. A retina slice preparation with whole cell patch recording techniques under voltage- and current-clamp conditions was used to assay the electrical properties of bipolar and amacrine cells. Amacrine cells were categorized into two basic forms: (i) transient amacrine cells that respond to a step of light with a burst of spikes only at the transitions of the step; and (ii) sustained amacrine cells that respond with continuous spiking during the entire light step. The two cell types each had a characteristic morphology: transient amacrine cells possessed wide dendritic fields (chi = 375 microns), while sustained cells had much more narrowly confined dendritic fields (chi = 85 microns). Whole cell voltage-gated currents of the transient and sustained cell types were not significantly different. Both cell types had spikes that were sensitive to tetradotoxin (TTX, 0.3 microM) with voltage deflections of up to 100 mV. Light-evoked excitatory synaptic currents relaxed rapidly in transient neurons (tau 1/2 = 100 ms) and more slowly in sustained neurons (tau 1/2 = 1.2 s). EPSCs in both cells reversed near 0 mV. Rapid application of glutamate or kainate elicited rapidly desensitizing ionic currents (tau 1/2 = 85 ms) followed by a slowly desensitizing component. Cyclothiazide, a drug that eliminates rapid desensitization, lengthened the time course of the glutamate gated current from tau 1/2 = 85 ms to about 3 s, and the relaxation kinetics of the glutamatergic EPSC from tau 1/2 = 85 ms to about 1.0 s. These data suggest that a key determinant in forming transient versus sustained responses in amacrine cells of vertebrate retina is the differences in their excitatory, glutamatergic synaptic inputs, and that rapid desensitization of glutamate receptors plays a role in converting the presynaptic signal associated with sustained glutamate release into a postsynaptic, transient signal at the ribbon synapse.

Action Potentials↗

Calcium-dependent inactivation and depletion of synaptic cleft calcium ions combine to regulate rod calcium currents under physiological conditions.

L-type Ca2+ currents (I(Ca)) in rod photoreceptors exhibit Ca2+-dependent inactivation. Perforated-patch whole-cell recordings were obtained from isolated rods of the tiger salamander using 1.8 mm Ca2+ in the bathing medium to determine the extent of Ca2+-dependent inactivation of I(Ca) with physiological [Ca2+] and endogenous buffering. I(Ca) was measured with voltage ramps applied before and after 5-s steps to -40, -30, -20, or -10 mV. Long depolarizing steps in isolated rods produced inactivation of I(Ca) ranging from 15% at -40 mV to > 80% at -10 mV. Because, in addition to Ca2+-dependent inactivation, depletion of synaptic cleft Ca2+ accompanying activation of I(Ca) can reduce presynaptic I(Ca) at calycal synapses, we investigated whether a similar mechanism worked at the invaginating rod synapse. Rods from retinal slices with intact synapses were compared with isolated rods in which synaptic cleft depletion is absent. I(Ca) was more strongly depressed by depolarization of rods in retinal slices, with ICa reduced by 47% following voltage steps to -40 mV. The depression of currents by depolarization was also greater for rods from retinal slices than isolated rods when Ca2+ was replaced with Ba2+ to reduce Ca2+-dependent inactivation. The stronger depolarization-evoked inhibition of I(Ca) in retinal slices compared to isolated rods probably reflects depletion of synaptic cleft Ca2+ arising from sustained Ca2+ influx. Inactivation of I(Ca) exhibited slow onset and recovery. These findings suggest that Ca2+-dependent inactivation and depletion of synaptic cleft Ca2+ may combine to regulate I(Ca) in response to light-evoked changes in rod membrane potential.

Ambystoma↗

Edmund Randerath (1899-1961): experimental proof for the glomerular origin of proteinuria.

A century ago, Edmund Randerath (1899-1961), who was one of the pioneers in nephrology that provided indirect experimental proof for the glomerular origin of proteinuria, was born. In the first decades of this century, the concept prevailed that "nephrosis" was a process of primary tubular cell degeneration. In contrast to prevailing opinion, he interpreted these changes to be the result of the uptake and storage of serum proteins after they had been filtered in the glomerulus. Edmund Randerath proved the glomerular origin of proteinuria by astute experiments in amphibia. In the salamander, an intraperitoneal injection of albumin provoked the supposedly "degenerative" changes of tubular epithelial cells in only those nephrons that drained the coelomic cavity and were devoid of glomeruli, but not in those nephrons that were closed and attached to glomeruli. This observation provided incontrovertible evidence that the presence of serum proteins in tubular fluid was a prerequisite for the development of the tubular epithelial cell changes typically seen in nephrotic patients.

Animals↗

Apoptosis in regenerating and denervated, nonregenerating urodele forelimbs.

Denervated limbs of larval salamanders fail to regenerate if amputated and, unlike adult limbs, undergo regression. The cellular basis of the tissue loss is poorly understood. We used TUNEL staining of larval axolotl limbs fixed and sectioned at intervals after bilateral amputation and unilateral denervation to investigate the role of apoptosis during normal limb regeneration and denervated limb regression. In the first week after amputation a small percentage of apoptotic cells was found in both innervated and denervated limbs. During the second week the apoptotic index remained low in the mitotically active mesenchymal cells of the regenerating limbs, but increased twofold in the nondividing, dedifferentiated cells of the regressing limbs. TUNEL-positive cells resembling apoptotic bodies were restricted primarily to the dedifferentiated area beneath the wound epithelium, but were also present within the wound epithelium itself. Macrophages were identified immunohistochemically and were also found in increased numbers in distal areas of the denervated regressing limbs. The results suggest a role for apoptosis in the early phase of normal regeneration and indicate that denervated limb regression involves an increased rate of apoptosis and removal of apoptotic bodies by macrophages and the wound epithelium.

Animals↗